Electrophoresis, as used in the fields of biology, molecular biology, biochemistry, clinical chemistry and medicine, is an analytical technique for separating and identifying biologically important molecules in a sample. Applications include the determination of a sample's homogeneality, the determination of molecular weights of proteins and nucleic acids, the mapping of nucleic acid primary structures, i.e. DNA and RNA sequence analyses, and the definition of phenotypic variance of a protein at the molecular level. A variety of techniques have been developed in order to accomplish these tasks. However, all these techniques rely on the fact that each molecular species has a unique combination of size, shape, charge, density, and sub-unit structure. Each of the electrophoretic techniques uses one or more of these parameters to cause varying degrees of molecular separation via the migration of the molecular species under the influence of an electric field.
One technique for separation of molecules is referred to as capillary zone electrophoresis. This technique uses a capillary tube which is filled with a conductive fluid, or buffer solution. A small amount of a sample is introduced at one end of the capillary tube, whereafter a high potential difference is applied across the ends of the tube. Differences in the electrophoretic mobilities of different molecules cause the constituents of the sample to emerge separated at the outlet end of the capillary tube. Capillary zone electrophoresis is described in greater detail in U.S. Pat. No. 4,842,701 to Smith et al.
One concern in electrophoresis separation by use of an electromotive force is maintaining the temperature of the capillary tube within a desired range. A typical potential difference across the ends of the capillary tube is in the range of 20,000 to 30,000 volts. Current through the capillary tube causes heating of the solution and the capillary tube. Heating affects, and may completely destroy, results of the quantitative and qualitative analysis. The biological sample within the buffer solution may be damaged or destroyed by excessive heat. Moreover, boiling may occur and cause a recurring loss of electrical conduction, and therefore, produce a lower or diminished current flow and a breakdown of the electrophoresis process.
One method of controlling the temperature of electrophoresis process is to use a heat sink for the capillary tube. U.S. Pat. No. 4,708,782 to Andresen et al. teaches use of a heat sink for the capillary. Likewise, U.S. Pat. No. 4,705,616 to Andresen et al. teaches the heat sink and further provides a double-feed-line approach which allows the capillary electrophoresis probe tip to be continually wetted by the buffer so as to improve heat dissipation. U.S. Pat. No. 4,612,106 to Kromer et al. is related to gel slab electrophoresis, but that patent also teaches use of a heat sink as being critical to temperature control. A serpentine piping through the heat sink provides an air circulating system to augment heat dissipation.
Cooling methods other than use of a heat sink are known. In each case, it is important to address the problem of isolating the high voltage source associated with capillary zone electrophoresis. As noted above, a potential difference of 30,000 volts may be employed. Immersing the capillary tube in tap water to dissipate the heat is risky because tap water is conductive and, if the capillary tube were to form a crack or if the electrodes were not well isolated from the water, unsafe charging of the ungrounded water or shorting to grounded water could occur. De-ionized water may be a better choice, but any contamination may render the water conductive. A known solution is to use a cooled oily, non-conductive, perfluorinated fluid with a high dielectric strength to contact the capillary tube. The use of the specialized oily fluid, unfortunately, is expensive and sometimes cost- prohibitive. Moreover, the oily fluid causes difficulties in cleanup after completion of the electrophoresis process. Another method may be to achieve a degree of capillary zone electrophoresis cooling by use of air in contact with the capillary tube. The drawbacks are that still air does not sufficiently conduct heat away from the capillary tube, and that a flow of air along the length of the capillary tube creates temperature differentiation within the tube as the air is heated by continued flow along the length.
An object of the present invention is to provide an apparatus for electrophoretic separation using a capillary tube, wherein temperature control is provided in a manner which isolates heat dissipation to achieve a uniform cooling and which isolates the operational high voltage source.